1. Field of the Invention
This invention relates to non-invasive imaging methods and minimally invasive sensing methods for assessing the viability of implanted stem cells and for the early detection of the transformation of implanted stem cells into brain tumors.
2. Background of the Art
Studies have demonstrated that the symptoms of Parkinson's disease (PD) can be improved by transplanting dopaminergic (DA) stem cells into the brain of PD patients. A major obstacle to effective stem cell therapy of PD is that only a low percentage of cells implanted into the brain survive more than seven days (Bjorklund, A., “Neurobiology—Better Cells for Brain Repair,” Nature 363(6419), 414-415, (Apr. 1, 1993)). Many factors have been shown to influence long-term viability of DA stem cell implants, including the site of the implant, the specificity of donor tissue, and the techniques used in the preparation of the cells to be implanted. One potential factor which may account for the high attrition rate of implanted stem cells is a lack of nutritive support. Angiogenesis in cell implants has been shown to occur after approximately three days post-implantation, which suggests that most implanted cells die within the first 48 to 72 hours after implantation when they are dependent on local diffusion for oxygen and glucose; see, for example, Watts, C., et al., “The Development of Intracerebral Cell-Suspension Implants is Influenced by the Grafting Medium,” Cell Transplantation, 7(6), 573-583, (November-December 1998). The precise mechanism of survival of implanted cells until a vascular supply becomes established is not known, although it may be influenced by the oxygen tension of the local environment during the course of graft vascularization; see, for example, Stokes, B. T., et al., “Oxygen-Transport in Intraspinal Fetal Grafts—Graft Host Relations,” Experimental Neurology, 111(3), 312-323, (March 1991).
A method to monitor non-invasively the ongoing viability of the cell implant is needed, in particular to determine whether the cells are adequately perfused by the local microvasculature. U.S. Pat. No. 5,190,744 to Rocklage et al. discloses MRI methods for evaluating local and regional tissue perfusion based on first-pass tracking of a bolus of MRI contrast agent. U.S. Pat. No. 5,494,655 to Rocklage et al. additionally discloses MRI methods for evaluating local and regional tissue perfusion changes induced by administration of a vasodilatory or vasoconstrictive drug agent. U.S. Pat. No. 5,833,947 to Rocklage et al. further discloses MRI methods for evaluating local and regional tissue perfusion changes induced by surgical procedures, such as cell implants. U.S. patent application. Ser. No. 09/606,137, co-authored by two of the present authors, discloses methods for indicating viability of transplanted progenitor or stem cells based on imaging measurements of changes in blood flow in the region of the cell implant. Unlike the present invention, however, none of these patents disclose a method for quantitating the functional capillary density in the anatomic region of the cell implant, or for quantitatively determining the metabolic status of a population of living cells implanted into a tissue in a human body.
The clinical utility of cell therapy is further limited by the fact that the incidence of teratoma tumor formation from implanted embryonic stem cells remains high despite significant recent advances in implant methodology. Magnetic resonance (MR) methods have been used to investigate the relationship of tumor metabolism to blood flow and oxygenation, proliferation, and differentiation. Several reviews published in the medical literature have summarized the morphological, metabolic, and physiological characteristics of tumors and their relationship to 1H, 13C, and 31P measurements obtained by MR spectroscopy; see for example Wehrle, J. P., et al., “31P NMR-Spectroscopy of Tumors in vivo,” Cancer Biochemistry Biophysics, 8(3), 157-166, (1986) and Howe, F. A., et al., “Proton Spectroscopy in-vivo,” Magnetic Resonance Quarterly, 9(1), 31-59, (March 1993).
Tumor growth to a volume of about 1 cubic mm can occur without contiguous microvascular support, since all the essential nutrients and waste products can diffuse across this distance. However, blood vessels are essential for further tumor progression. Inadequate local blood flow and low concentrations of glucose and oxygen appear to influence the latency of expression of DNA damage. Blood flow also controls cellular environment and heat clearance, factors which are important in hyperthermic treatment of tumors. The sensitivity of cells to radiation depends significantly on the concentration of cellular oxygen. A non-invasive imaging method of monitoring blood flow and oxygenation during the post-transplant period, in conjunction with methods to modify these parameters, would increase the effectiveness of early detection of tumors and potentially improve treatment strategies during the early stages of tumor development following embryonic stem cell implants.
Tumor vascular supply is derived from normal vessels incorporated from the host tissue and new blood vessels stimulated by tumor angiogenesis factors; see Folkman, J., et al., “Angiogenic Factors,” Science, 235(4787), 442-447, (Jan. 23, 1987). Neovascular development is characterized by various structural abnormalities, including an absence of smooth muscle cells, collapsed vessels due to increased tissue pressure, stasis, large sinusoidal structures, arteriovenous shunts, and thrombosis; see Jain, R. K., “Determinants of Tumor Blood Flow—a Review,” Cancer Research, 48(10), 2641-2658, (May 15, 1988).
If neovascularization associated with new tumors cannot match the rapid proliferation of tumor cells, the result is a reduced and inhomogeneous supply of blood, substrates, and oxygen leading to hypoxia, anoxia, and ultimately cell death. Surviving cells generally are located at distances of 150 microns or less from the nearest blood vessel; see Thomlinson, R. H., et al., British Journal of Cancer, 9, 539-549, (1955). However, cellular debris, fatty acids, proteins, and nucleic acid fragments present in necrotic areas can also interfere with mitochondrial functioning of cells in adjacent perfused areas; see Falk, P., “Differences in Vascular Pattern between the Spontaneous and the Transplanted C3H Mouse Mammary-Carcinoma,” European Journal of Cancer & Clinical Oncology, 18(2), 155-165, 1982. The composition of tumor interstitial fluid is similar to normal interstitial fluid, except for high concentrations of lactate (10-30 mM), and a very low content of free glucose (0-2 mM). Tumors also have elevated interstitial pressure, which has been attributed to the absence of functioning lymphatics, the high filtration coefficient and vascular permeability of tumor blood vessels, and the rapid proliferation of cells in confined spaces; see Less, J. R., et al., “Interstitial Hypertension in Human Tumors. 4. Interstitial Hypertension in Human Breast and Colorectal Tumors,” Cancer Research, 52(22), 6371-6374, (Nov. 15, 1992).
By comparison with other methods, MR is capable of measuring blood flow and oxygenation non-invasively, either indirectly by evaluating metabolism or by using contrast agents. 1H MR spectra of brain tumors show increased lactate and total choline and reduced N-acetyl aspartate (NAA) levels compared to normal brain spectra; see Negendank, W., “Studies of Human Tumors by MRS—A Review,” NMR in Biomedicine, 5(5), 303-324, (September-October 1992). The high levels of lactate are consistent with high glycolytic rates and poor blood flow associated with tumors. The high levels of total choline may be due to increased membrane degradation or turnover, since the choline compounds observed in 1H MR spectra are both membrane precursors and breakdown products. Hypoxia is known to result in membrane breakdown and the release of free fatty acids.
Compared to other brain tumors, teratomas almost always exhibit calcific, lipomatous, or cystic foci, making their MRI diagnosis relatively easy. For example, the high signal intensity of a teratoma on a T1-weighted (TR 600/TE 24 ms) sequence is suggestive of fat, an impression that can be verified by the loss of signal intensity over two echoes of a long TR sequence. A cystic focus in a teratoma exhibits a chemical shift artifact which can be readily appreciated on MR imaging. While the prior art discloses imaging methods for detecting and diagnosing diseases of the central nervous system, including brain tumors, imaging methods which provide for early detection of teratomas that originate from implanted stem cells have not been previously disclosed.
U.S. Pat. No. 6,319,682 to Hochman discloses optical detection techniques for the assessment of physiologic state and metabolic viability of biological materials, including cells. An express purpose of the Hochman patent is high throughput screening of candidate agents and conditions to evaluate their suitability as diagnostic or therapeutic agents. However, unlike the present invention, Hochman does not disclose a method means for assessing the metabolic viability of implanted stem cells and for the early detection of their transformation into teratomas.
U.S. Pat. No. 6,497,872 to Weiss et al. discloses methods of transplanting multipotent neural stem cell progeny to a host by obtaining a population of cells derived from mammalian neural tissue containing at least one multipotent neural stem cell; culturing the neural stem cell in a culture medium containing one or more growth factors which induce multipotent neural stem cell proliferation; inducing proliferation of the multipotent neural stem cell to produce neural stem cell progeny; and transplanting the multipotent neural stem cell progeny to the host. Also provided in the patent to Weiss et al. are methods of transplanting neural stem cell progeny to a host by obtaining an in vitro cell culture containing CNS neural stem cells and transplanting the stem cells into the host.
U.S. Pat. No. 6,503,478 to Chaiken et al. discloses methods and materials for obtaining spatially resolved images of specific types of tissues. The method for imaging tissue comprises administering to the tissue a deuterated imaging agent and performing spectroscopy. Electromagnetic radiation, such as a near infrared laser beam, is directed to a tissue of interest. When used in combination with a light collection system, it is possible to map out a specific volume of tissue, obtaining information regarding the distribution of specific endogenous chemical species. In some embodiments disclosed by Chaiken et al., specific imaging agents are employed to impart contrast between chemically different types of tissues.
U.S. Pat. No. 6,521,210 to Ohkawa discloses a method for imaging a malignancy in a patient, in situ, but requires feeding the patient a nutrient that is enriched with the isotope 13C. MRI techniques are then used on the patient with rf energy that is tuned to the nuclear resonance of 13C. An image of selected tissue in the patient is thereby created, and is evaluated for any concentrations of 13C that will delineate a malignancy. A subsequent MRI procedure may be performed to determine the efficacy of any intervening treatment, or to determine a growth rate for the malignancy However, unlike the present invention, the prior art does not disclose a method for assessing the metabolic activity and perfusion status of implanted stem cells as a practical and reliable MR imaging means for early detection of the transformation of implanted cells into brain tumors.